The goal of this project is to understand the mechanisms by which mutations in the Inverted Formin 2 gene INF2 cause focal segmental glomerulosclerosis (FSGS) in humans. We and others have identified numerous FSGS-segregating mutations, demonstrating that INF2 mutations are the most common autosomal dominant form of FSGS. INF2 is unique for a formin family member in that it accelerates both actin polymerization and depolymerization. Formins autoinhibit their activity by an interaction between two domains, the N-terminal DID (diaphanous inhibitory domain) and the C-terminal DAD (diaphanous autoregulatory domain), and are generally activated by binding of a small GTPase to the N-terminus near the DID. INF2 has two major splice variants, one associated with the endoplasmic reticulum (ER) via a C-terminal prenyl group, and another lacking the prenyl group. In the first period of this grant, we have made significant progress towards understanding how mutations in INF2 cause human kidney disease. We have defined roles for INF2 in cells and organelles. We have shown that INF2 binds to and modulates the activity of the diaphanous formin (Dia) family of RhoA effectors. We have developed mouse and zebrafish models for the in vivo study of INF2 and its associated mutations. In contrast to essentially all other actin regulatory proteins, INF2-DID mutations are a relatively common form of human FSGS, suggesting that INF2-DID possesses unique and non-redundant functions in the podocyte. Our long-term goal is to understand these functions and, ultimately, exploit them for therapeutic benefit. Now, we aim to: (1) Define the specific biochemical effects of representative disease-causing mutants. We will test the hypothesis that the 30+ known INF2 mutations can each lead to disease through four possible defects: 1. Destabilization of INF2 protein structure, leading to instability/degradation; 2. Disruption of auto- inhibition of INF2 through the DID/DAD interaction; 3. Altered regulation of Dia family formins through interaction of INF2-DID with Dia-DAD; 4. Disruption of other inter-molecular interactions. (2) Define the effects of FSGS mutations on INF2 cellular function. We will: 1. Define INF2 and Dia protein localization and isoform expression in podocytes; 2. Determine cellular effects of FSGS mutations on INF2 and Dia function; 3. Assess the influence of Dia proteins on FSGS mutant effects (3) Correlate in vitro and cell-based studies with in vivo models (zebrafish, mouse). We will: 1. Use a zebrafish INF2 knockdown model to compare the direct phenotypic effects of a range of different perturbations in INF2 (knockdown, transgenesis) in response to changes in RhoA/Rac/Cdc42 signaling; 2. Use the results from these studies to guide experiments in mouse point mutant (knockin) and knockout models, investigating the molecular mechanisms of injury in a mammalian kidney.